1. Field of the Invention
The present invention generally relates to the field of methods of gene knock-out with interstrand binding between a specific probe and its target sequence. More particularly, the present invention relates to the field of improved methods for knocking out specific gene expression by covalently bonding formation between a gene transcript and its anti-sense probe.
2. Description of the Prior Art
The following references are pertinent to this invention:
1. Falletta et.al., "Phase 1 Evaluation of Diaziquone in Childhood Cancer", Investigational New Drugs 8: 167-170 (1990). PA1 2. Hartley et.al., "DNA Cross-linking and Sequence Selectivity of Aziridinylbenzoquinones", Biocldenmistry 30: 11719-11724 (1991). PA1 3. Hampson et.al., "Chemical Crosslinking Subtraction; A New Method for the Generation of subtractive hybridization probes", Nucleic Acids Res. 20: 2899 (1992). PA1 4. Kimler et.al., "Combination of Aziridinylbenzoquinone and Cis-platinum with Radiation Therapy in the 9L Rat Brain Tumor Model", International Journal of Radiation Oncology, Biology, Physics 26: 445-450 (1993). PA1 5. Majumdar et.al., "Stepwise Mechanism of HIV Reverse Transcriptase: Primer Function of Phosphorothioate Oligodeoxynuclcotide", Biochemistry 28: 1340-1346 (1989). PA1 6. Matsukura et.al., "Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate against rev (art/trs) in chronically infected cells", Proc. Natl. Acad. Sci. 86: 4244-4248 (1989). PA1 7. Sambrook et.al., "Molecular Cloning, 2nd Edition", Cold Spring Harbor Laboratory Press (1989). PA1 8. Solomons ct.al., "Organic Chemistry, 6th Edition", John Wiley & Sons Press (1996). PA1 9. Tan et.al., "Phase 1 Study of Aziridinylbenzoquinone in Children with Cancer", Cancer Research 44: 831-835 (1984). PA1 10. U.S. Pat. No. 4,599,303 issued to Yabusaki et.al. PA1 11. U.S. Pat. No. 4,689,320 issued to Kaji et.al. PA1 12. U.S. Pat. No. 4,806,463 issued to Goodchild et.al. PA1 13. U.S. Pat. No. 5,023,243 issued to Tullis et.al. PA1 14. U.S. Pat. No. 5,030,557 issued to Hogan et.al. PA1 15. U.S. Pat. No. 5,110,802 issued to Contin et.al. PA1 16. U.S. Pat. No. 5,589,339 issued to Hampson et.al. PA1 17. U.S. Pat. No. 5,591,575 issued to Hampson et.al. PA1 18. U.S. Pat. No. 5,739,309 issued to Dattagupta et.al. PA1 a. providing a first strand of nucleotide sequences as complementary probes to a targeted gene transcript, wherein said first strand of nucleotide sequences is single-stranded and carboxylated in the nucleotide base structures of said first strand of nucleotide sequences; PA1 b. preserving said first strand of nucleotide sequences in a delivery vector, wherein said delivery vector transports said first strand of nucleotide sequences into cells; PA1 c. contacting said first strand of nucleotide sequences with a second strand of gene transcripts in said cells, wherein said second strand of gene transcripts contains natural amino-groups in the nucleotide base structures of said second strand of gene transcripts; and PA1 d. permitting said first strand of nucleotide sequences and said second strand of gene transcripts to form double-stranded hybrid duplexes comprising covalently bonding between the carboxyl-groups of said first strand and the amino-groups of said second strand. PA1 a. an amino-blocking reagent which makes said first strand of nucleotide probes single-stranded; PA1 b. a carboxylating reagent which generates said carboxyl-groups on the nucleotide base structures of said first strand probes; and PA1 c. a delivery vector which permits hybridization of said first strand probes and said second strand gene transcripts in said cells to form said covalently bonded hybrid duplexes.
Anti-sense sequences of certain gene transcript have been widely used as probes in gene knock-out systems to detect specific gene functions as well as to perform clinical therapy for many years. Based on the hydrogen-bonding of natural nucleotide sequences, many kinds of anti-sense probes were designed to interfere normal functions of certain gene transcript (mRNA) by increasing the binding stability of probe-mRNA hybrids. These anti-sense probes include sequence-specific "helper" oligonucleotides (U.S. Pat. No. 5,030,557 by Hogan et.al.; U.S. Pat. No. 5,023,243 by Tullis et.al.; U.S. Pat. No. 4,689,320 by Kaji et.al.), multiple-targeting oligonucleotides (U.S. Pat. No. 4,806,463 by Goodchild et.al.), methylphosphorate-linked oligonucleotides (U.S. Pat. No. 5,110,802 by Contin et.al.) and phosphorothioate-linked oligonucleotides (Majumdar et.al., Biochemistry 28: 1340 (1989); Matsukura et.al., Proc. Natl. Acad. Sci. 86: 4244 (1989); U.S. Pat. No. 5,739,309 by Dattagupta et.al.). Some of special chemical linkages used in aforementioned anti-sense probes are designed for reducing nuclease digestion of probes and increasing sequence-specific targeting. However, all these anti-sense technologies specifically bind to targeted sequences through hydrogen-bonding affinity, resulting a requirement of exceedingly high concentration treatments for biological effects.
On the other hand, the ability to form covalently bonding between two nucleotide sequences has permitted a fully complete subtraction of undesired sequences through hybridizative binding. Unlike hydrogen-bonding formed between natural nucleotide sequences, because the covalent bond is one of the strongest and most heat-stable interactions between molecules, covalently bonding of two nucleotide sequences can sustain most harsh treatments or conditions, such as denaturing, salting, desalting, enzyme digestion and some redox reaction. Based on such feature, some methods have been developed either to perform anti-sense chemotherapy for killing cancer cells as well as viruses or to detect unique gene expressions with cross-linking chemicals by which unwanted homologues were covalently eliminated. One of the most commonly used cross-linking chemicals to accomplish such sequence-selective elimination is aziridinylbenzoquinone (AZQ)-class agent (Hartley et.al., Biochemistry 30: 11719-11724 (1991)), involving a cross-linking between guanine (G) and cytosine (C).
In clinics, AZQ-class agents have been tested as a chemotherapy drug for treating several cancers, such as brain tumors in rat (Kimler et.al., International Journal of Radiation Oncology, Biology, Physics 26: 445-450 (1993)) and phase I childhood cancers in human (Falletta et.al., Investigational New Drugs 8: 167-170 (1990); Tan et.al., Cancer Research 44: 831-835 (1984)). By enhancing the binding, stability of genomic double-stranding conformations, the AZQ-clke molecules w ere expected to reduce the replication of the cancer cells. However, the nonspecific cross-linking feature of AZQ-like molecules also causes significant toxicity to normal cells. Thus, the use of AZQ-like molecules in an anti-sense gene therapy seems unpractical now. Since the lack of sequence-specific targeting capability is an unsolved problem in vivo, in vitro methods become the only feasible way to apply AZQ-cike cross-linking molecules for subtracting unwanted genes.
Prior art attempts at in vitro subtraction of targeted sequences with covalently bonding, such as U.S. Pat. No. 5,589,339 and U.S. Pat. No. 5,591,575 to Hampson et.al., uses te cross-linking agents to covalently subtract unwanted sequences from compared libraries. Unfortunately, although the AZQ-like cross-linking molecules successfully achieve the completion of homologue subtraction, such subtraction only occurs in test tubes with single-stranded oligonucleotide samples. Other prior art attempts at sequence-targeting, such as U.S. Pat. No. 4,599,303 to Yabusaki et.al., uses azide-like cross-linking molecules to detect isolated targets which are unavailable in living cells. Therefore, in addition to the significantly cytotoxic nature of cross-linking molecules, the disadvantages of these methods exclude the possibility to subtract specific gene expression from living cells.
In summary, it is desirable to have a simple, specific and nontoxic gene knock-out method for increasing binding efficiency of an anti-sense probe to its targeted gene transcript, of which the results may contribute to an investigation of new gene functions, a diagnosis for inherent problems, or an anti-sense therapy for diseases.